Familial amyotrophic lateral sclerosis (FALS; Lou Gehrig's Disease; motor neuron disease) has missense mutations in the coding regions in the Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene. Several studies suggested that the dominant, gain-of-function of FALS Cu,Zn-SOD mutant plays a key role in the pathogenesis of FALS. We investigated the nature of this gain-of-function of FALS Cu,Zn-SOD mutants. Two different mechanisms were suggested for the gains-of-function: (1) the enhancement of the free radical generating function of Cu,Zn-SOD in addition to the normal dismutation activity, which we found previously; (2) the enhancement of the ability to catalyze nitration of tyrosine residues in proteins. To compare these enzyme activities in the mutant and the wild-type enzymes, we cloned the wild-type and two mutants (G93A and A4V) cDNA of human Cu,Zn-SOD, overexpressed them in insect cells (sf9), purified the proteins, and studied their enzymic activities. Our results showed that the wild-type, G93A, and A4V enzymes have identical dismutation activity. However, the free radical generating function of the G93A and A4V mutants, as measured by spin trapping and electron paramagnetic resonance spectroscopic methods, is enhanced relative to that of the wild-type enzyme (wild-type<G93A<A4V), particularly at lower hydrogen peroxide concentrations. For the nitration of tyrosine residues, no such enhanced activity was found with these FALS mutants. The enhancement of the free radical generating function is due to the decrease in the Km value for hydrogen peroxide (wild-type>G93A>A4V), while the k(cat) is identical for these enzymes. The fact that the A4V mutant has the lowest Km is correlated to the clinical severity observed with the A4V patients, if FALS is associated with a differential gain of the free radical generating function of the Cu,Zn-SOD mutant.